The long-term goal of this project is to understand how vitamin A signaling is modulated by orphan nuclear receptors belonging to the TR2 and TR4 family. Previous studies focused on the repressive mechanisms of TR2 that include i) directly recruiting co-repressors like histone deacetylases (HDACs), receptor interacting protein 140 (RIP140) and SMRT (an active mechanism), and ii) competing with RA receptors (RARs) and retinoid X receptor (RXR) for DNA binding (a passive mechanism). By extending from these conclusions and based upon recent studies, this renewal proposal focuses on novel ligand-independent signaling pathways that can modify the property and activity of TR2 and TR4 for the regulation of RAR|32, cyclin D1 and apoE genes. Three hypotheses will be tested: i) the biological activity of TR2 and TR4 can be modulated by protein modification (biochemical factors) and their interaction with coregulators (kinetic factors), ii) the physiologically relevant receptor activity is manifested through their interaction with, or recruitment of, specific coregulators onto the regulatory region of the target gene (dynamic factors), and iii) the ligand- independently activated receptor complex can contribute to chromatin remodeling of target gene to activate transcription. Aim I will address the first and second hypotheses by examining the mechanisms of ligand-independent modulation of receptor activity elicited through protein modifications (using a proteomic approach) that affect: i) the biochemical nature of receptors, ii) the general property of receptors, iii) receptor-coregulator interaction kinetics and iv) dynamics of TR2 and TR4 coregulatory complex on target genes. Aim II will address the third hypothesis in physiologically relevant cell cultures by manipulating TR2 and TR4, and determining the effects of their modification on target genes. The biological effects to be examined include the formation of coregulatory complexes and alteration in chromatin conformation (remodeling) or histone modification on the target gene promoters (RAR|32, cyclin Dl and apoE) and their biological activities in P19 cell cycle progression as well as transcription efficiency of target genes. Results from both in vitro (aim 1) and in vivo (aim 2) systems will be integrated to construct a comprehensive overview of the mechanisms underlying the modulation of vitamin A signaling pathways by these orphan receptors, specifically with respect to signals generated from protein modifications of receptors. [unreadable] [unreadable]